Production of 3-hydroxypropionic acid in recombinant organisms

ABSTRACT

The production of 3-hydroxypropionic acid (3-HP) from glycerol in a bacterial host is described. 3-HP is a useful feedstock for the production of polymeric materials. The genetic engineering of a bacterial host with two enzymes is sufficient to enable production of 3-HP. One enzyme is a glycerol dehydratase and the other is an aldehyde dehydrogenase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional PatentApplication Ser. No. 60/151,440 filed Aug. 30, 1999.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The research project which gave rise to the invention described in thispatent application was supported by EPA grant R824726-01. The UnitedStates Government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

The technology of genetic engineering allows the transfer of genetictraits between species and permits, in particular, the transfer ofenzymes from one species to others. These techniques have first reachedcommercialization in connection with high-value added products such aspharmaceuticals. The techniques of genetic engineering are equallyapplicable and cost effective when applied to genes and enzymes whichcan be used to make basic chemical feedstocks.

A metabolic pathway of interest exists in the bacteria Klebsiellapneumoniae, which has the ability to biologically produce3-hydroxypropionaldehyde from glycerol. Native microorganisms have theability to produce 1,3-propanediol from glycerol as well. Commercialinterests are exploring the production of 1,3-propanediol from glycerolor glucose, in recombinant organisms which have been engineered toexpress the enzymes necessary for 1,3-propanediol production from otherorganisms.

3-hydroxypropionic acid CAS registry Number [503-66-2] (abbreviated as3-HP) is a three carbon non-chiral organic molecule. The IUPACnomenclature name for this molecule is propionic acid 3-hydroxy. It isalso known as 3-hydroxypropionate, β-hydroxpropionic acid,β-hydroxypropionate, 3-hydroxypropionic acid, 3-hydroxypropanoate,hydracrylic acid, ethylene lactic acid, β-lactic acid and 2-deoxyglcericacid. Applications of 3-HP include the manufacture of absorbableprosthetic devices and surgical sutures, incorporation intobeta-lactams, production of acrylic acid, formation oftrifluromethylated alcohols or diols, polyhydroxyalkonates, andco-polymers with lactic acid. 3-HP for commercial use is now commonlyproduced by organic chemical syntheses. The 3-HP produced and sold bythese methods is relatively expensive, and it would be cost prohibitiveto use it for the production of monomers for polymer production. Asdiscussed below, some organisms are known to produce 3-HP. However,there is not yet available a catalog of genes from these organisms andthus the ability to synthesize 3-HP using the enzymes nativelyresponsible for the synthesis of that molecule in the native hosts whichproduce it does not now exist.

In addition to its commercial utility, 3-HP it is found in a number ofbiological processes, notably including many naturally occurringbio-polymers. Poly(3-hydroxybutyrate) (PHB) is the most abundant memberof the microbial polyesters which contain hydroxy monomers termedpolyhydroxyalkonates (PHAs). PHB has utility as a biodegradablethermoplastic material and the material was first produced industriallyin 1982.

The majority of published research on PHA's that contain 3-HP hasconcentrated on two bacterial sources: Ralstonia eutropha (“Alcaligeneseutrophus”) and Pseudomonas oleovorans. Both Ralstonia eutropha andPseudomonas oleovorans are able to grow on a nitrogen free mediacontaining 3-hydroxy -propionic acid, 1,5-pentanediol or1,7-heptanediol. When 3-HP is the major hydroxy-acid added to the growthmedia, poly(3-hydroxybutyrate-co-3-hydroxypropionic acid) is formedcontaining 7 mol % 3-hydroxypropionic acid. These cells also store 3 mol%, 3-hydroxypropionic acid poly(3-butyrate-co-3-hydroxypropionic acid).

Recombinant systems have been used to create PHAs. An E. coil strainengineered to express PHA synthase from either Ralstonia eutropha orZoolgoea ramigera produced poly(3-hydroxypropionic acid) when feed1,3-propanediol. Skraly, F. A. “Polyhydroxyalkonates Produced byRecombinant E. coli.” Poster at Engineering Foundation Conference:Metabolic Engineering II, 1998. An E. coli strain that expressed PHAsynthase (MBX820), when provided with the genes encoding glyceroldehydratase and 1,3-propanediol dehydratase from K. pneumonia, and4-hydroxybutyral-CoA transferase from Clostridium kluyveri, synthesizedPHB from glucose.

Glycerol dehydratase, found in the bacterial pathway for the conversionof glycerol to 1,3-propanediol, catalyzes the conversion of glycerol to3-hydroxypropionaldehyde and water. This enzyme has been found in anumber of bacteria including strains of Citrobacter, Klebsiella,Lactobacillus, Entrobacter and Clostridium. In the 1,3-propanediolpathway a second enzyme 1,3-propanediol oxido-reductase (EC 1.1.202)reduces 3-hydroxypropanaldehyde to 1,3-propanediol in a NADH dependantreaction. The pathway for the conversion of glycerol to 1,3-propanediolhas been expressed in E. coli. Tong et al., Applied and EnvironmentalMicrobiology 57 (12)3541-3546. The genes responsible for the productionof 1,3-propanediol were cloned from the dha regulon of Klebsiellapneumoniae. Glycerol is transported into the cell by the glycerolfacilitator, and then converted into 3-hydroxy-propionaldehyde by acoenzyme B₁₂-dependent dehydratase. E. coli lacks a native dha regulon,consequently E. coli cannot grow aerobically on glycerol without anexogenous electron acceptor such as nitrate or fumarate.

Aldehyde dehydrogenases are enzymes that catalyze the oxidation ofaldehydes to carboxylic acids. The genes encoding non-specific aldehydedehydrogenases have been identified in a wide variety of organisms e.g.;ALDH2 from Homo sapiens, ALD4 from Saccharomyces cerevisiae, and from E.coli both aldA and aldB, to name a few. These enzymes are classified byco-factor usage, most require either AND⁺, or NADP⁺ and some will useeither co-factor. The genes singled out for mention here are able to acton a number of different aldehydes and it likely that they may be ableto oxidize 3-hydroxy-propionaldehyde to 3-hydroxypropionic acid.

BRIEF SUMMARY OF THE INVENTION

The present invention is intended to permit the creation of arecombinant microbial host which is capable of synthesizing 3-HP from astarting material of glycerol or glucose. The glycerol or glucose isconverted to 3-hydroxypropionicaldehyde (abbreviated as 3-HPA) which isthen converted to 3-HP. This process requires the so-called dhaB genefrom Klebsiella pneumoniae which encodes the enzyme glycerol dehydrataseany one of four different aldehyde dehydrogenase genes to convert 3-HPAto 3-HP. The four aldehyde dehydrogenase genes used were aldA from thebacterium E. coli, ALDH2 from humans, ALD4 from the yeast Saccharomycescerevisiae, and aldB from E coli. The yeast gene appeared to give thebest results.

It is an object of the present invention to provide a genetic constructwhich encodes glycerol dehydratase and aldehyde dehydrogenase enzymesnecessary for the production of 3-hydroxypropionic acid from glycerol.

It is also an object of the present invention to provide a method forthe production of 3-hydroxypropionic acid from glycerol.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiment thereof and from theclaims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Not applicable.

DETAILED DESCRIPTION OF THE INVENTION

It is disclosed here that it is possible to introduce into a bacterialhost genes encoding two enzymes and thus confer upon that host theability to produce 3-HP from glycerol. The two necessary enzymes areglycerol dehydratase and aldehyde dehydrogenase. It is here reportedthat the two enzymes are both necessary and sufficient to enable astrain of a suitable host, such as a competent E. coli strain, to make3-HP from glycerol. An exemplary gene encoding a glycerol dehydratase isknown, the dhaB gene from Klebsiella pneumoniae, sequenced and renderedconvenient to use. Several exemplary aldehyde dehydrogenases are known,and their sequences are presented here. From this information, itbecomes practical to confer upon a bacterial host the ability to convertglycerol into 3-HP in a commercially reasonable manner.

It was not apparent before the completion of the work described herethat these two diverse enzymes could be produced in a common host toproduce the ability to make 3-HP. There are many known aldehydedehydrogenase enzymes and genes, and the enzymes are known to havevarying substrate specificities and efficiencies. There was notevidence, prior to the work described here, that the aldehydedehydrogenase enzyme would work on the 3-hydroxypropionicaldehyde(3-HPA) substrate to create 3-HP. Without that knowledge, there was nodata from which to predict the effectiveness of the 3-HP productionstudies described below. An additional uncertainty arises from the factthat the intermediate aldehyde, 3-HPA, is toxic to many bacterial hostand thus the survival of the host is dependent upon the relative ratesof enzymatic production and conversion of the aldehyde intermediate tonon-toxic 3-HP.

A difficulty in the realization of the production of 3-HP desired hereis that ribosome binding sites from non-native hosts are oftenineffectual and lead to poor protein production and that many non-nativepromoters are often poorly transcribed and a bar to high proteinexpression. However, the inventors also recognized that a non-nativepromoter that is known to be very active and is inducible by theaddition of a small molecule unrelated to the pathway being expressed isoften a very efficient way to express and regulate the levels of enzymesexpressed in hosts such as E. coli. To achieve high levels of regulatedgene expression plasmids were constructed which placed the expression ofall exogenous genes necessary for the production of 3-hydroxypropionicacid from glycerol under the regulation of the trc promoter. The trcpromoter, is efficient, not native to E. coli, and inducible by theaddition of IPTG.

The present specification describes a genetic construct for use in theproduction of 3-hydroxypropionic acid from glycerol. The geneticconstruct includes exemplary DNA sequences coding for the expression ofa glycerol dehydratase and a DNA sequence coding for aldehydedehydrogenase. The set of exemplary sequences necessary for theexpression of glycerol dehydratase is collectively referred to as“dhaB”. The set of sequences necessary for the expression of aldehydedehydrogenase includes any one of four different genes which provedefficacious. The individual aldehyde dehydrogenase sequences referred toindividually as ALDH4, ALD2, aldA and aldB.

Producing 3-hydroxypropionic Acid in a Foreign Host

In the work described below, the enzymes necessary for the production of3-hydroxypropionic acid from glycerol in E. coli were expressed underthe regulation of the trc promoter, a non-native promoter inducible bythe addition of IPTG. The glycerol dehydratase was encoded by the dhaBgene from Klebsiella pneumoniae, the aldehyde dehydrogenases used wasany one of four different genes (ALDH2 from Homo sapiens, ALD4 from S.cerevisiae, aldB from E. coli or aldA from E. coli). Expression of thesegenes coding for glycerol dehydratase and any one of the genes encodingan aldehyde dehydrogenases was sufficient to enable the construct toproduce 3-HP when the fermentation media was supplemented with glycerol.In all of these constructs, the dhaB gene was downstream from the geneencoding the aldehyde dehydrogenase used, and expression of both geneswas regulated by the trc promoter. This order, however, is not requiredand the order of the gens on a construct and the use of multipleconstructs is possible.

In a minimal genetic construct made based on the data presented here,the only genetic elements present that would be necessary are thestructural genes dhaB and an aldehyde dehydrogenase gene encoding aprotein that efficiently catalyzes the oxidation of3-hydroxypropionaldehyde to 3-hydroxypropionic acid, and non-nativepromoter sequences specifically selected to give the type of induciblecontrol most appropriate for the context of the process in which theconstruct is to be used. Extraneous pieces of DNA, whether retained inthe construct or added from other DNA sequences, would not necessarilybe detrimental to effective 3-HP synthesis by the host organism, butwould not be needed. Each sequence to be translated would necessarily bepreceded by a ribosome binding site, functional in the selected host sothat the messenger RNA(s) coding for the proteins of interest could betranslated by ribosomes. Terminator sequences immediately downstream ofeach translated unit would also be necessary in some organisms,particularly in eukaryotes. The construct could be part of anautonomously replicating sequence, such as a plasmid or phage vector, orcould be integrated into the genome of the host.

The structural genes and appropriate promoter(s) could be isolated bythe use of restriction enzymes, by the polymerase chain reaction (PCR),by chemical synthesis of the appropriate oligonucleotides, or by othermethods apparent to those skilled in the art or molecular biology. Thepromoter(s) would be derived from genomic DNA of other organism or fromartificial genetic constructs containing promoters. Appropriate promoterfragments would be ligated into the construct upstream of the structuralgenes in any one of several possible arrangements.

The aldehyde dehydrogenase expressed would have: high specific activitytowards 3-hydroxypropionaldehyde; be very stable in the host it isexpressed in; be readily over expressed in the selected host; not beinhibited by either the substrates necessary for the reaction or theproducts formed by the reaction; be fully active under the fermentationconditions most favorable for the production of 3-hydroxypropionic acidand be able to use either NAD⁺ or NADP⁺.

One possible arrangement is the true operon, where one promoter is usedto direct transcription in one direction of all necessary Open ReadingFrames (ORFs). The entire message is then contained in one messengerRNA. The advantages of the operon are that it is relatively easy toconstruct, since only one promoter is needed; that is it is relativelysimple to replace the promoter with another promoter if that would bedesirable later; and that it assures that the two genes are under thesame regulation. The main disadvantage of the operon scheme is that thelevels of the expression of the two genes cannot be variedindependently. If it is found that the genes, for optimal3-hydroxypropionic acid synthesis, should be expressed at differentlevels, the operon in most cases cannot be used to realize this.

Another possible arrangement is the multiple-promoter scheme. Two ormore promoters, with the same or distinct regulatory behavior, could beused to direct transcription of the genes. For example, one promotercould be used to direct transcription of dhaB and one to directtranscription of the gene encoding the appropriate aldehydedehydrogenases. Because the genes theoretically can be transcribed andtranslated separately, a great number of combinations of multiplepromoters is possible. Additionally, it would be most desirable toprevent the promoters from interfering with one another. This could beachieved either by placing two promoters into the construct such thatthey direct transcription in opposite directions, or by insertingtranscriptional terminator sequences downstream of each separatelytranscribed unit. The main advantage of the multiple-promoter constructis that it permits independent regulation of as many distinct units asdesired, which could be important. The disadvantages are that it wouldbe more difficult to construct; more difficult to amend later; and moredifficult to effectively regulate, since multiple changes infermentation conditions would need to be introduced and might render theperformance of the fermentation somewhat less predicable.

In any construct, the promoter sequence(s) used should be functional inthe selected host organism and preferably provide sufficienttranscription of the genes comprising the glycerol to 3-hydroxypropionicacid pathway to enable the construct to be adequately active in thathost. The promoter sequence(s) used would also effect regulation oftranscription of the genes enabling the glycerol to 3-HP pathway to beadequately active under the fermentation conditions employed for 3-HPproduction, and preferably they would be inducible, such that expressionof the genes could be modulated by the inclusion in, or exclusion from,the fermentation of a certain agents or conditions.

A plausible example of the use of such a construct follows: onepromoter, which induced by the addition of an inexpensive chemical (theinducer) to the medium, could control transcription of both the dhaBgene and the gene encoding the appropriate aldehyde dehydrogenase. Thecells would be permitted to grow in the absence of the inducer untilthey accumulated to a predetermined level. The inducer would then beadded to the fermentation and nutritional changes commensurate with thealtered metabolism would be made to the medium as well. The cells wouldthen be permitted to utilize the substrate(s) provided for 3-HPproduction (and additional biomass production if desired). After thecells could no longer use substrate to produce 3-HP, the fermentationwould be stopped and the 3-HP recovered.

Genetic Sequences

To express glycerol dehydratase and a suitable aldehyde dehydrogenase,the two enzymes necessary for the production of 3-hydroxypropionic acidfrom glycerol, it is required that the DNA sequences containing theglycerol dehydratase and aldehyde dehydrogenase coding sequences becombined with at least a promoter sequence (preferably a non-nativepromoter although some native promoter activity may be present). Anexemplary method of construction is described in the example below. Toensure that the present specification is enabling, the full sequences ofthe coding regions of genes for these enzymes is presented here.

Sequences 1, 3, 5 and 7 present different native genomic sequences forgenes encoding aldehyde dehydrogenases.

SEQ ID NO:1 contains the full native DNA sequence encoding the ALD4enzyme from Saccharomyces cerevisiae. The amino acid sequence of theprotein is presented as SEQ ID NO:2.

SEQ ID NO:3 includes the DNA sequence for the human ALDH2 gene, againincluding the full protein coding region. The amino acid sequence forthis human alcohol dehydrogenase is presented in SEQ ID NO:4.

SEQ ID NO:5 and 7 respectively present the full coding sequences fromthe E. coli genes aldA and aldB, both of which encode alcoholdehydrogenases. The amino acid sequences for the proteins encoded by thegenes are presented in SEQ ID NO:6 and 8 respectively.

SEQ ID NO:9 contains the native genomic DNA sequence for the dhaB genefrom the dha regulon of Klebisiella pneumoniae. The coding sequences forthis complex regulon produces five polypeptides, which are presented asSEQ ID NOS:10 through 13, which together provide the activity of theglycerol dehydratase enzyme.

Each of these coding sequences can be used to make genetic constructsfor the expression of the appropriate enzymes in a heterologous hosts.In making genetic constructs for expression of the genes in such hosts,it is contemplated that heterologous promoters will be joined to thecoding sequences for the enzymes, but all that it required is that thepromoters be effective for the hosts in which the genes are to beexpressed. It is also contemplated and envisioned that significantvariations in DNA sequence are possible from the native DNA codingsequences presented here. As is well known in the art, due to thedegeneracy of the genetic code, many different DNA sequences can encodethe expression of the same protein. So, when this document uses languagespecifying a DNA sequence encoding a protein, it is intended toencompass any DNA sequence which can be used to express that proteineven if different from the genomic sequences presented here. It is alsocontemplated that conservative changes in the amino acid sequences ofthe proteins specified here can be made without departing from thepresent invention. In particular, deletions, additions and substitutionsof one or more amino acids in a protein sequence can almost always bemade without changing protein functionality. When the name of a proteinis sued here, it is intended to be equally applicable to both such minorchanges in amino acid sequence and to allelic variations in nativeprotein sequence as occurs within the species named as well as otherclosely related species.

It is possible that many of the above DNA sequences could be truncatedand still express a protein that has the same enzymatic properties. Oneskilled in the art of molecular biology would appreciate that minordeletions, additions and mutations may not change the attributes of thedesignated base pair sequences; many of the nucleotide of the designatedbase pair sequences are probably not essential for their uniquefunction. To determine whether or not an altered sequence or sequenceshas sufficient homology with the designated base pairs to functionidentically, one would simply create the candidate mutation, deletion oralteration and create a gene construct including the altered sequencetogether with promoter and termination sequences. This gene constructcould be tested as, described below, for the production of 3-HP fromglycerol.

Certain DNA primers were used to isolate or clone the genomic DNAsequences used in the experiments described below. While the sequenceinformation presented here is sufficient to enable the construction ofexpression plasmids incorporating the genes identified here, in order toredundantly enable the use of these genes, primers which may be used toisolated the genes from their native hosts are described below.

The primers aldA_L (SEQ ID NO:14), and aldA_R (SEQ ID NO:15), were usedto amplify the 1513 bp aldA fragment from genomic E. coli DNA (strainMG1655, a gift from the Genetic Stock Center, New Haven, Conn.). The gelpurified PCR fragment containing a DNA sequence coding for theexpression of aldehyde dehydrogenase was inserted into NcoI-XhoI site ofpSE380 (Invitrogen, San Diego, Calif.) to give pPFS3. The resultingplasmid contained aldA under the control of the trc promoter. Thisconstruct allowed for high-level expression of the aldA gene from E.coli under regulation of the trc promoter. Unless indicated otherwiseall molecular biology and plasmid constructions were done in E. coli AG1(Stratagene, La Jolla, Calif.).

The primers aldB_L (SEQ ID NO:20) and aldB_R (SEQ ID NO:21), were usedto amplify the 1574 bp aldB fragment from genomic E. coli DNA (strainMG1655). The resulting PCR converted the TGA stop codon into a TAA stopcodon. The gel-purified PCR fragment containing the DNA sequencesufficiently coding for the expression of aldehyde dehydrogenase wasinserted into the KpnI-SacI site of pSE380 to give pPFS12.

The primers ALD4_L (SEQ ID NO:16), and ALD4_R (SEQ ID NO:17), were usedto amplify the 1595 bp ALD4 fragment from S. cerevisiae DNA (strainYPH500). The gel-purified fragment containing a DNA sequence coding forthe expression of aldehyde dehydrogenase was inserted into the KpnI-SacIsite of pPFS3 to give pPFS8. The resulting plasmid contained mature ALD4under control of the trc promoter.

The primers ALDH2_L (SEQ ID NO:18), and ALDH2_R (SEQ ID NO:19), wereused to amplify the 1541 bp ALDH2 fragment from pT7-7::ALDH2, a giftfrom H. Weiner (Purdue University, West Lafayette, Ind.). The gelpurified PCR fragment containing a DNA sequence sufficiently homologousto base pairs 22 to 1524, inclusive of SEQ ID NO:3 so as to code for theexpression of aldehyde dehydrogenase was inserted in to the KpnI-SacIsite of pSE380 to give pPFS7. This sequence was moved from pPFS7 intothe KpnI-SacI site of pPFS3 to give pPFS9. The resulting plasmidcontained mature ALDH2 under the control of the trc promoter.

The primers pTRC_L (SEQ ID NO:22), and pTRC_R )SEQ ID NO:23), were usedto amplify the 540 bp fragment from pSE380. The gel purified PCRfragment was inserted into the HpaI-KpnI site of pPFS3 to give pPFS13.The resulting plasmid deleted the “native” ribosome binding site ofpSE380 and a NcoI site (which contained an extraneous ATG start codonupstream of the cloned genes). The KpnI-SacI fragments of pPFS8, pPFS9,and pPSF12 were inserted into the KpnI-SacI site of pPFS13 to givepPFS14, pPFS15, and pPFS16, respectively.

Assay for Production of 3-HP

The efficacy of changes made as contemplated herein can be checked bythe following tests. To test for the production of 3-HP, fermentationproducts can be quantified with a Waters Alliance Integrity HPLC system(Milford, Mass.) equipped with a refractive index detector, a photodiodearray detector, and an Aminex HPX-87H (Bio-Rad, Hercules, Calif.)organic acids column. The mobile phase should be 0.01 N sulfuric acidsolution (pH 2.0) at a flow rate of 0.5 mL/min. The column temperatureshould be set to 40° C. Compounds can be identified by determining ifthey co-elute with authentic standards. Prior to analysis, all samplesshould be filtered through 0.45 μM pore size membrane. (Gelman Sciences,Ann Arbor, Mich.). The fractions of the fermentation products collectedusing HPLC should be analyzed on a Varian Star 3400 CX,gas-chromatograph coupled to a Varian Saturn 3 mass spectrometer (GC-MS)(Walnut Creek, Calif.).

Assay for Enzyme Activity

Aldehyde dehydrogenase activity can be determined by measuring thereduction of β-NAD⁺ at 25° C. with 3-hydroxypropionaldehyde as asubstrate. All buffers should contain 1 mM ethylenediaminetetraaceticacid (EDTA), 0.1 mM Pefabloc SC (Boehringer Mannheim, Indianapolis,Ind.) and 1 mM Tris (carboxyethyl) phosphine hydrochloride (TCEP-HCL).For ALD4, the solution should contain 100 mM Tris HCL Buffer (pH 8.0),100 mM KCl. For ALDH2 the solution should contained 100 mM sodiumpyrophosphate (pH 9.0). For AldA and AldB, the solution should contain20 mM sodium glycine (pH 9.5). A total of 3.0 mL of buffer should beadded to quartz cuvettes and allowed to equilibrate to assaytemperature. From 5 to 20 μL of cell extract should be added andbackground activity recorded after the addition of β-NAD⁺ to a finalconcentration of 0.67 mM. The reaction should be started by the additionof substrate (either acetaldehyde, propionaldehyde, or3-hydroxypropionaldehyde) to a final concentration of 2 mM. Assaymixtures should be stirred with micro-stirrers during the assays.

For aldehyde dehydrogenase activity assays, one unit is defined as thereduction of 1.0 μM of β-AND⁺ per minute at 25° C. These reactions canbe monitored by following the change in absorbence at 340 nm (A₃₄₀) at25° C. on a Varain Carry-1 Bio spectrophotometer (Sugar Land, Tex.).Total protein concentrations in the cell extracts can be determinedusing the Bradford assay method (Bio-Rad, Hercules, Calif.) with bovineserum albumin as the standard.

EXAMPLES ps Plasmid Constructions

Klebsiella pneumoniae expresses glycerol dehydratase, an enzyme thatcatalyzes the conversion of glycerol to 3-hydroxypropionaldehyde, (dhaB)and 1,3-propanediol oxidoreductase an enzyme that catalyzes theconversion of 3-hydroxypropionaldehyde to 1,3-propanediol respectively(the gene product from dhaT). A plasmid encoding these two genes wascreated and expressed in E. coli (plasmid pTC53). The dhaT gene wasdeleted from pTC53 to create pMH34. The resulting plasmid stillcontained the DNA sequence complementary to base pairs 330 to 2153inclusion of SEQ ID NO:9, the complement of base pairs 2166 to 2591,inclusive, of SEQ ID NO:9, and the complement of base pairs 3191 to4858, inclusive, of SEQ ID NO:9, so as to code for the expression ofglycerol dehydratase. The fragment of DNA encoding these sequences wasexcised from pMH34 by cutting it with SalI-XbaI, and the resultingfragment was gel purified (the purified fragment was gift from M.Hoffman of the University of Wisconsin—Madison). This DNA fragment wasinserted into the SalI-XbaI site of pPFS13 to give pPFS17.

The resulting plasmid contained both the aldA and dhaB genes under thecontrol of the trc promoter. Similarity, the gel-purified SalI-XbaIfragment from pMH34 was inserted into the SalI-XbaI sites of pPFS14,pPFS15, and pPFS16 to give pPFS18, pPFS19, and pPFS20, respectively.These plasmids contained ALD4, ALDH2, and aldB, respectively, as well asdhaB under the control of the trc promoter; in all of the constructs thedhaB gene were downstream of the gene encoding the aldehydedehydrogenase.

Expression in E. coli

The efficacy of E. coli as a platform for the production of 3-HP fromgrowth on glucose has been examined using a mathematical model developedfor this purpose. The model was executed in two different ways assumingthe conversion of one mole of glucose under either anaerobic or aerobicconditions either directly to 3-HP or to the production of 3-HP and ATP.The optimum yield under anaerobic conditions is 1 mole of 3-HP and 1mole of lactate. The more realistic yield under anaerobic conditions is0.5 moles of 3-HP, 1.5 moles of lactate and 1 mole of ATP. The optimumyield under aerobic conditions is 1.9 moles of 3-HP and 0.3 moles ofCO₂. The realistic yield under aerobic conditions is 1.85 moles of 3-HP,0.35 moles of CO₂ and 1 mole of ATP.

The effect of 3-HP concentration on E. coli strain MG1655 growth wasmeasured. Cells were grown on standard media with and without theaddition of up to 80 g/L of 3-HP. The best fit of these datademonstrated that 3-HP was only 1.4 times as inhibitory as lactic acidon the growth of E. coli. It is possible to economically produce lacticacid using E. coli, since 3-HP is only 1.4 times more inhibitory thanlactic acid, it should be possible to use E. coli as a host for thecommercial production of 3-HP.

Media and Growth Conditions

The standard media contained the following per liter: 6 g Na₂HPO₄, 3 gKH₂PO₄, 1 g NH₄Cl, 0.5 g NaCl, 3 mg CaCl₂, 5 g yeast extract (DifcoLaboratories, Detroit, Mich.) and 2 mM MgSO₄. When necessary to retainplasmids ampicillin (100 mg/mL) was added to the media.Isopropyl-β-thiogalactopyranoside (IPTG) was added in varying amounts toinduce gene expression. All fermentations were carried out in anincubator-shaker at 37 C and 200 rpm. Anaerobic fermentations werecarried out in 500-mL anaerobic flasks with 300 mL of working volume.Inocula for fermentations were grown overnight in Luria-Bertani mediumsupplemented with ampicillin is necessary. The 300-mL fermentations wereinoculated with 1.5 mL of the overnight culture. For enzyme assays,fermentations were incubated for 24 hours.

Over Expression of Aldehyde Dehydrogenase in E. coli.

Cells were harvested by centrifugation at 3000×g for 10 minutes at 4° C.with a Beckman (Fullerton, Calif.) model J2-21 centrifuge. Cell pelletswere washed twice in 100 mM potassium phosphate buffer at pH 7.2 andre-suspended in appropriate assay resuspension buffer equal to 5× of thevolume of the wet cell mass. The cells were homogenized using a Frenchpressure cell. The homogenate was centrifuged at 40000×g for 30 minutes.The supernatant was dialyzed against the appropriate resuspension bufferusing 10000 molecular weight cut-off pleated dialysis tubing (Pierce,Rockford, Ill.) at 4° C. Dialysis buffer was changed after 2 hours, and4 hours, and dialysis was stopped after being allowed to proceedovernight.

E. coli AG1 cells transfected with the plasmids constructed to expressthe aldA, ALD4, ALDH2, or aldB genes were grown in 500-mL anaerobicflasks. Twelve hours after the fermentations were inoculated IPTG wasadded to induce enzyme expression. The cells were allowed to grow for anadditional 12 hours then harvested and lysed as discussed above. Thesoluble fraction of the lysate was assayed for aldehyde dehydrogenaseactivity using the substrate 3-hydroxypropionicaldehyde in the bufferappropriate for the particular enzyme expressed The plasmid, aldehydedehydrogenase expressed and specific activity measured (U/mg of protein)were as follows: pPFS13, aldA, 0.2; pPFS14, ALD4, 0.5, pPFS15, ALDH2,0.3; and pPFS16, aldB.0.1. The control, E. coli strain AG1 harboringplasmid pSE380, encoded no exogenous aldehyde dehydrogenase activity andit had no detectable activity with 3-HP as substrate. It is clear fromthe activity assays that all four aldehyde dehydrogenases were expressedin E. coli. The aldehyde dehydrogenase cloned from Saccharomycescerevisiae (ADH4) had the highest activity when 3-hydroxypropionaldehydewas used as the substrate (0.5 units/mg of protein).

E. coli cells transformed with plasmids expressing: aldehydedehydrogenase; both aldehyde dehydrogenase and glycerol dehydratase, orneither gene; were grown and assayed for their ability to produce 3-HPfrom glycerol. The cells were grown on standard media supplemented with6 μM of Coenzyme B₁₂, under anaerobic conditions in the absence of light(to protect the integrity of the Coenzyme B₁₂ necessary for DhaBactivity). After 12 hours, IPTG was added to induce expression of thegenes under the trc promoter at the same time 5 g/L of glycerol wasadded. After 12 more hours of anaerobic fermentation the fermentationbroth was assayed for 3-HP by HPLC and GC, the plasmid, aldehydedehydrogenase gene expressed and g/L of 3-HP measured were as follows:pSF17, aldA, 0.031; pPSF18 ALD4, 0.173; and pPSF19, ALDH2, 0.061. Cellsexpressing dhaB but no exogenous aldehyde dehydrogenase genes (plasmidpMH34) produced 0.015 g/L of 3-HP. Cells expressing aldA, ALD4, ALDH2 oraldB but not dhaB (plasmids pPFS13, pPFS14, pPFS15, pPFS16,respectively) all produced less then 0.005 g/L of 3-HP when the mediathe cells were growing in was supplemented with 2.5 g/L of3-hydroxypropionaldehyde.

Other Hosts and Promoters

Applications of the 3-hydroxypropionic acid pathway such as the geneticconstructs of the present invention can easily be expressed in otherorganisms. The required genes would need to be placed under control ofan appropriate promoter or promoters. Some organism such as yeasts mayrequire transcription terminators to be placed after each transcribedunit. The knowledge of the present intention makes such amendmentspossible. Such a genetic construct would need to be part of a vectorthat could either replicate in the new host or integrate into thechromosome of the new host. Many such vectors are commercially availablefor expression in gram-negative and gram-positive bacteria, yeast,mammalian cells, insect cell, plant, etc. For example, to express the3-hydroxypropionic acid pathway in Rhodobacter capsulatus, one couldobtain vector pNH2 from the American Type Culture Collection (ATTC).This is a shuttle vector for use in R. capsulatus and E. coli. Organismssuch as Saccharomyces cerevisiae which can convert glucose to glycerolcould be used as a host, such a construct would enable the production of3-HP directly from glucose. Additionally, other substrates such as xylancould also be used given the selection of an appropriate host.

Stochiometric analysis shows that best stochiometric yield of 3-HPproduction in E. coli calculated on the basis of glucose consumed isobtained under aerobic conditions. Under aerobic condition CO₂ is theonly carbon-containing co-product, in particular the generation oflactic acid which occurs under anaerobic conditions is avoided.Production of 3-HP under these conditions could result in a moreeconomical recovery of 3-HP from the fermentation broth.

Alternatively, the dhaB gene and a gene encoding the appropriatealdehyde dehydrogenase could be cloned into the multiple cloning site ofthis vector in E. coli to facilitate construction, and then transformedinto R. capsulatus. The R. capsulatus nifH promoter, provided on theplasmid, could be used to direct the transcription in R. capsulatus ofthe genes placed into pNF2 in series with one promoter, or with twocopies of the nifH promoter. Expression of the genes in other organismswould require a procedure analogous to that presented here.

Alternative Aldehyde Dehydrogenases and Glycerol Dehydratases

Applications of the pathway for the production of 3-hydroxypropionicacid from glycerol can be made using other suitable aldehydedehydrogenases. To be functional in this pathway an aldehydedehydrogenase needs to be stable, readily expressed in the host ofchoice and have high enough activity towards 3-hydroxypropionaldehyde toenable it to make 3-HP. The knowledge of the present invention makessuch amendments possible. A program of directed evolution could beundertaken to select for suitable aldehyde dehydrogenases or they couldbe recovered from native sources, the genes encoding these enzymes inconjunction with a gene encoding an appropriate glycerol dehydrataseactivity, would then be made part of any of the constructs envisionedhere to produce 3-hydroxypropionic acid from glycerol.

A similar program of enzyme improvement including for example directedevolution could be carried out using the dhaB gene from Klebsiellapneumoniae as a starting point to obtain other variants of glyceroldehydratase that are superior in efficiency and stability to the formused in this invention. Alternatively, enzymes which catalyzes the samereaction may be isolated from others organisms and used in place of theKlebsiella pneumoniae glycerol dehydratase. Such enzymes may beespecially useful in alternative hosts wherein they may be more readilyexpressed, be more stable and more efficient under the fermentationconditions best suited to the growth of the construct and the productionand recovery of 3-HP.

1. A method for producing 3-hydroxypropionic acid comprising the stepsof providing in a fermenter a recombinant microorganism which carries agenetic construct which expresses the dhaB gene from Klebsiellapneumoniae and a gene for an aldehyde dehydrogenase, which are capableof catalyzing the production of 3-hydroxypropionic acid from glycerol;providing a source of glycerol or glucose for the recombinantmicroorganism, and fermenting the microorganism under conditions whichresult in the accumulation of 3-hydroxypropionic acid in solution in thefermenter.
 2. A method for producing 3-hydroxypropionic acid comprisingthe steps of providing in a fermenter a recombinant microorganism whichcarries a genetic construct which expresses the dhaB gene fromKlebsiella pneumoniae and a gene for an aldehyde dehydrogenase, whichare capable of catalyzing the production of 3-hydroxypropionic acid fromglycerol; providing a source of glycerol or glucose for the recombinantmicroorganism, and fermenting the microorganism under conditions whichresult in the accumulation of 3-hydroxypropionic acid wherein the genefor the aldehyde dehydrogenase is selected from the group consisting ofALDH2, ALD4, aldA and aldB.
 3. A method for producing 3-hydroxypropionicacid comprising the steps of providing in a fermenter a recombinantmicroorganism which carries a genetic construct which expresses the dhaBgene from Klebsiella pneumoniae and a gene for an aldehydedehydrogenase, which are capable of catalyzing the production of3-hydroxypropionic acid from glycerol; providing a source of glycerol orglucose for the recombinant microorganism, and fermenting themicroorganism under conditions which result in the accumulation of3-hydroxypropionic acid wherein the aldehyde dehydrogenase selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ IDNO:8.
 4. A recombinant E. coli host comprising in its inheritablegenetic materials foreign dhaB gene from Klebsiella pneumoniae and agene for aldehyde dehydrogenase, such that the host is capable ofproducing 3-hydroxypropionic acid from glycerol.
 5. A recombinant E.coli host comprising in its inheritable genetic materials the dhaB genefrom Klebsiella pneumoniae and the ald4 gene from Saccharomycetescervisiae, such that the host is capable of producing 3-hydroxypropionicfrom glycerol.
 6. A bacterial host comprising in its inheritable geneticmaterial a genetic construction encoding for the expression of the dhaBgene from Klebsiella pneumoniae and an aldehyde dehydrogenase enzyme,such that the bacterial host is capable of converting glycerol to3-hydroxypropionic acid.
 7. A bacterial host comprising in itsinheritable genetic material a genetic construction encoding on of aglycerol dehydratase enzyme, the amino acid sequence of which areselected from SEQ IDS NO:10, 11, 12 and 13, and an aldehydedehydrogenase enzyme, such that the bacterial host is capable ofconverting glycerol to 3-hydroxypropionic acid wherein the aldehydedehydrogenase is selected from the group consisting of SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6 and SEQ ID NO:8.
 8. A bacterial host comprising inits inheritable genetic material a genetic construction encoding for theexpression of a glycerol dehydratase enzyme, the amino acid sequence ofwhich are selected from SEQ IDS NO:10, 11, 12 and 13, and an aldehydedehydrogenase enzyme, such that the bacterial host is capable ofconverting glycerol to 3-hydroxypropionic acid wherein the gene for thealdehyde dehydrogenase is selected from the group consisting of ALDH2,ALDA4, aldA and aldB.